Abstract
The polymerase chain reaction (PCR) has become an essential tool in molecular biology for the rapid amplification and analysis of DNA (1). The technique requires the use of oligonucleotide primers that bind to the DNA at a specific region of interest. The region is subsequently copied by enzymatic polymerization of deoxynucleotides. Among its many applications, PCR has found use in fields where the quality of results is of primary importance, such as in disease diagnosis (2) or forensic DNA analysis (3). In these cases, PCR amplification from very small quantities of DNA is often required (4,5), requiring high-reaction efficiency. Additionally, the consequences of ambiguous or false results in assays are far-reaching, and it is therefore essential that the PCR is highly specific. In order to obtain such high-performance standards the components of the PCR must be carefully controlled, particularly with respect to deoxynucleoside triphosphates (dNTPs) and oligonucleotide primers.
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Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., and Erlich H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Schochetman G., Ou C.-Y., and Jones W. K. (1988) Polymerase chain reaction. J. Infect. Dis. 158, 1154–1157.
Reynolds R., Sensabaugh G., and Blake E. (1991) Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction. Anal. Chem. 63, 2–15.
Higuchi R., von Beroldingen C. H., Sensabaugh G. F., and Erlich H. A. (1988) DNA typing from single hairs. Nature 332, 543–545.
Jeffreys A. J., Wilson V., Newman R., and Keyte J. (1988) Amplification of human minisatellites: Towards DNA fingerprinting of single cells. Nucleic Acids Res. 16, 10,953–10,971.
Takigiku R. and Schneider R. E. (1991) Reproducibility and quantitation of separation for ribonucleoside triphosphates and deoxyribonucleoside triphosphates by capillary zone electrophoresis. J. Chromatogr. A 559, 247–256.
O’Neill K., Shao X., Zhao Z., Malik A., and Lee M. L. (1994) Capillary electrophoresis of nucleotides on ucon-coated fused silica columns. Anal. Biochem. 222, 185–189.
Uhrova M., Deyl M., and Suchanek M. (1996) Separation of common nucleotides, mono-, di-and triphosphates by capillary electrophoresis. J. Chromatogr. B-Biomedical Applications 681, 99–105.
Geldart S. E. and Brown P. R. (1998) Analysis of nucleotides by capillary electrophoresis. J. Chromatogr. A 828, 317–336.
Cohen A. S., Najarian D. R., Paulus A., Guttman A., Smith J. A., and Karger B. L. (1988) Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis. Proc. Natl. Acad. Sci. USA 85, 9660–9663.
Manabe T., Chen N., Terabe S., Yohda M., and Endo I. (1994) Effects of linear polyacrylamide concentrations and applied voltages on the separation of oligonucleotides and DNA sequencing fragments by capillary electrophoresis. Anal. Chem. 66, 4243–4252.
Khan K., Van Schepdael A., and Hoogmartens J. (1996) Capillary electrophoresis of oligonucleotides using a replaceable sieving buffer with low viscosity-grade hydroxyethyl cellulose. J. Chromatogr. A. 742, 267–274.
Chiari M., Damin F., Melis A., and Consonni R. (1998) Separation of oligonucleotides and DNA fragments by capillary electrophoresis in dynamically and permanently coated capillaries, using a copolymer of acrylamide and beta-D-glucopyranoside as a new low viscosity matrix with a high sieving capacity. Electrophoresis 19, 3154–3159.
Devaney J. M. and Marino M. A. (2001) Purification methods for preparing polymerase chain reaction products for capillary electrophoresis analysis, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson K. R. and Cheng J., eds.), Humana Press Totowa, NJ, pp. 43–49.
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Pearce, M.J., Watson, N.D. (2001). Quality Control of Nucleotides and Primers for PCR. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology, vol 162. Humana Press. https://doi.org/10.1385/1-59259-055-1:347
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DOI: https://doi.org/10.1385/1-59259-055-1:347
Publisher Name: Humana Press
Print ISBN: 978-0-89603-779-3
Online ISBN: 978-1-59259-055-1
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